Recently, interest has emerged in label-free optical affinity-based biosensors, which allow study of bio-organisms without fluorescence or radiolabels, and thus dramatically simplify assays. Typically, affinity-based biosensors detect the presence of a target molecule by selective binding to a capture probe. For optical biosensors, binding translates into a change in optical properties, e.g., the complex refractive index or luminescence.
Optical detection methods based on complex refractive index transduction include interferometry in micro and nanofabricated devices, including porous thin films, Bragg reflectors, and microcavities, all of which require an optical measurement system with large beams and sensing areas (about 1 mm2), (see E. Chow, A. Grot, L. W. Mirkarimi, M. Sigalas, and G. Girolami, “Ultracompact biochemical sensor built with two-dimensional photonic crystal microcavity”, Optics Letters 29, 1093 (2004); L. L. Chan, B. T. Cunningham, P. Y. Li, D. Puff, “Self-referenced assay method for photonic crystal biosensors: Application to small molecule analytes”, Sens. Actuators B 120, 392 (2007); V. S.-Y. Lin, K. Motesharei, K. Motesharei, K.-P. S. Dancil, M. J. Sailor, and M. R. Ghadiri, Science 278, 840 (1997); F. Morhard, J. Pipper, R. Dahint, and M. Grunze, Sens. Actuators B 70, 232 (2000); M. Loncar, A. Scherer, and Y. Qiu, Appl. Phys. Lett. 82, 4648 (2003)).
Within the optical detection methods, photonic crystals constitute an emerging alternative technology due to their powerful light-confinement abilities which would enable local, and sensitive, refractive index measurements.
Extensive work has been performed during the last fifteen years to build and investigate photonic crystals, the optical analogues to electronic semiconductors. In semiconductors, electrons propagate in a periodic potential, which originates from the atomic lattice. This modifies the dispersion of free electrons and opens a band gap in the energy diagram, as shown in FIGS. 1A-1C.
In particular, FIGS. 1A-1C show electron dispersion in semiconductors. FIG. 1A shows a periodic lattice for silicon. FIG. 1B shows the induced periodic potential affecting the allowed electron energy states and shows Schrödinger's equation describing the quantum mechanical properties of electrons in a crystalline solid. FIG. 1C shows how solutions of the equations result in a band gap diagram with two allowed energy bands (valence band and conduction band) separated by a forbidden band (also called an electronic band gap).
Photonic crystals are materials built present a periodic variation of refractive index. With periodicity being of the same order of magnitude as the wavelength of the electromagnetic (EM) waves, these structures exhibit band gaps for photons, as indicated in FIGS. 2A-2C where photon dispersion in a 1D photonic crystal is shown. In particular, FIG. 2A shows the 1D periodic permittivity distribution, FIG. 2B shows Maxwell's equation describing the electromagnetic properties of photons in a medium of periodic refractive index, and FIG. 2C shows how solutions of the equation result in the opening of a forbidden band (also called photonic band gap) for the energy states of the photons.
Most of these devices are designed with opto-electronic applications in mind, and despite a recent step in the bio-sensing direction with blind 1D structures (see Schmidt, B., Alemeida, V., Manolataou, C., Prebel S., & Lipson, M., “Nanocavity in a silicon waveguide for ultrasensitive detection”, Appl. Phys. Lett. 85, 4854 (2004)) and non-specific chemical detection with blind 2D crystals, no selective chemical or biological detection has ever been reported with a 2D photonic platform (see the previously mentioned paper and also Levine, M. J. et al., “Zero-mode waveguides for single molecule analysis at high concentration”, Science, 299 (2003)).
The ability to manipulate photonic band gaps in the crystals by design offers the possibility of engineering highly resonant structures, and therefore high-Q microcavities, which makes photonic crystals attractive candidates for ultra compact, highly sensitive assays. Over a few μm2 sensing area, a few fL amount of sample analyte could be studied, providing the backbone for a very dense platform with single organism detection limit (lab-on-chip).
The various schemes and diagrams of FIG. 3 show a 1D photonic bio-sensing platform designed by Fauchet et al. (see M. R. Lee, and P. M. Fauchet, “Nanoscale microcavity sensor for single particle detection”, Optics Lett. 32, 3284 (2007)—S. Chan, S. R. Horner, P. M. Fauchet, & B. L. Miller, “Identification of Gram negative bacteria using nanoscale silicon microcavities”, J. Am. Chem. Soc. 123, 11797 (2001)).
The top scheme of FIG. 3 describes the device layout in which a 1D photonic structure is electrochemically etched on a silicon wafer. Layers of porous silicon with alternating high and low porosities constitute distributed Bragg reflectors (DBRs) around a luminescent central layer, also called a cavity. The entire assembly rests on the silicon substrate. The data shown in the four center diagrams of FIG. 3 corresponds to the luminescence of a series of cavities filtered by the surrounding DBRs and collected on the top of the device.
The darker lines of the two upper center diagrams are data collected after functionalization of the device with TWCP (tetratryptophan ter-cyclo pentane), a molecule that selectively binds lipid A, present in the viral coat of Gram(−) bacteria. The lighter lines of the two upper center diagrams are data collected after exposure of the functionalized device to Gram(−) bacteria (right) and Gram(+) bacteria (left). The lines of the two lower diagrams represent the difference between the darker and lighter lines discussed above and allow measuring of the spectral shift in photonic band gap resulting from the increase of refractive index in the DBRs upon binding of bacteria. The data is summarized in the bottom table of FIG. 3, indicating that no shift occurred upon exposure to Gram(+) bacteria while a 3-4 nm shift occurred upon exposure to 2 μg of Gram(−) bacteria.
Although the device presented in FIG. 3 can be used as a chemically functionalized 1D photonic crystal for bio-organism detection, the device presented in FIG. 3 requires the binding of a minimum of 2 μg of bacteria (thousands of organisms) to generate a positive signal. Indeed, the detection limit for a porous silicon crystal is inherently high because transduction is generated by a change of effective refractive index that has to occur across the entire volume of the crystal.
Functionalized silicon membranes were fabricated by electrochemistry and their ability demonstrated to selectively capture simulated bio-organisms. A photonic membrane can be defined as a photonic crystal formed of a periodic array of through-holes fabricated in a free-standing membrane waveguide, where the refractive index of the membrane material is larger than the refractive index of the surrounding air or liquid. A photonic membrane provides strong confinement of light both along and perpendicular to the plane of the membrane. In particular, FIG. 4 shows an SEM picture (top view in the background and cross section in the center) of a silicon membrane with 2 μm pores prepared by electrochemistry. This device was chemically functionalized with biotin and selective capture of streptavidin-functionalized beads (see central sphere in the bottom inset) was demonstrated, (see Létant, S. E., Hart, B. R., van Buuren, A. W. & Terminello, L. J., “Functionalized silicon membranes for selective bio-organism capture”, Nature Materials 2, 391 (2003)).
In order to add chemical specificity to size selectivity, nanoporous silicon devices were etched on pre-patterned silicon substrates and covalently functionalized with proteins (see Létant, S. E., Hart, B. R., Kane, S. R., Hadi, M., Shields, S. M. & Reynolds, J. G., “Enzyme immobilization on porous silicon surfaces”, Adv. Mat. 16, 689 (2004) and Hart, B. R., Létant S. E. et al., “New method for attachment of biomolecules to porous silicon”, Chem. Comm. 3, 322 (2003)). See also U.S. Pat. No. 7,155,076, incorporated herein by reference in its entirety.
The ability of the functionalized membranes to capture simulated bio-organisms was then successfully tested (as shown in FIG. 4 and in the related paper and patent mentioned above).